Project Summary Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease, but the molecular mechanisms driving the disease remain poorly understood. HCM displays a vast phenotypic heterogeneity, but is characterized by left ventricular hypertrophy unexplained by secondary causes, asymmetric, and typically accompanied by a preserved or increased ejection fraction. In addition, the cardiomyocytes often display hypertrophy and disarray in HCM. The clinical presentation can run the gamut from a relatively benign course to sudden cardiac death. HCM has primarily been thought of as a disease of the sarcomere with mutations in more than a dozen sarcomere-associated proteins correlated with HCM. However, up to about 70% of HCM cases do not have disease causing sarcomere mutations. Furthermore, the pleiotropic effects that are observed in both myocyte and non-myocyte populations in HCM are not easily explained by these mutations. To understand the underlying cause of HCM, we have chosen an unbiased approach to determine the pathogenic alterations at single cell resolution. At Tufts Medical Center, we have one of the largest HCM referral centers, performing approximately 100 septal myectomy surgeries per year to alleviate symptoms of obstructive HCM, a severe form of the disease. We propose to identify pathological pathways activated in human myectomy samples at single cell resolution. Our specific aims are: 1. Identify genes that are differentially expressed in different cardiomyocyte populations in human myectomy samples using single nuclei RNA-Seq 2. Develop a mouse model that recapitulates the human HCM phenotype 3. Characterize the role of RARRES1, a top candidate from our preliminary data, plays in hypertrophy, fatty acid metabolism, and autophagy in HCM. We will generate nuclei suspensions from myectomy tissue and use commercial systems to isolate thousands of individual nuclei for RNA-Seq. We will use CRISPR/Cas9 to target MYBPC3 to generate a mouse model that phenocopies human HCM to study drug targets and therapies of HCM. Top candidates from these studies will be further analyzed using mouse neonatal cardiomyocytes. The goal of these studies is to gain a better understanding of the underlying pathogenesis of HCM. This approach is innovative in that it uses an advanced single cell transcriptomics approach on a unique clinical resource available at our center. To the best of our knowledge, this approach has not been done before. The results from this study will not only lead to a better understanding of the pathology of this disease, but will uncover new potential therapeutic targets and treatments for patients.